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microRNA profiling of FFPE Sample Types

Archiving histological tissue samples by formalin-fixing and embedding in paraffin blocks has been a routine practice for medical and research institutions for many years and large collections of these samples now exist throughout the world. Estimates of sample numbers range from 300-400 million in the US alone covering virtually all known diseases. This number may be growing by tens of millions of samples per year.¹

What makes these samples of particular interest is the rich patient data and medical history associated with each sample. Because the samples are so well annotated, they are proving to be a valuable source of information for retrospective clinical studies of human diseases.

Initially, it was unclear if the nucleic acids would remain in sufficient condition to provide robust analytical results due to the adverse effects of formalin fixation. But studies have shown that although degradation to large RNA can occur, in general, small RNAs are unaffected by the fixation process². Indeed there are now a multitude of commercially available kits and methods for extracting microRNA from FFPE prepared tissue samples.³

Furthermore, it has been demonstrated in multiple studies that miRNA is minimally affected by FFPE treatment and microRNA extracted from FFPE samples provides reliable expression levels by direct comparison with frozen tissue samples.^4,5,6,7,8 MicroRNA expression data was found to be reliable for FFPE samples stored for up to 30 years.⁹

Example Data: FFPE vs Frozen Sample Expression Results

Kits for extraction of microRNA from FFPE prepared tissue samples:

RecoverAll Total Nucleic Acid Isolation Kit for FFPE – Ambion

miRNeasy FFPE Kit (50) – Qiagen

FFPE RNA Purification Kit – Norgen

Wang F, Wang L, Briggs C, Sicinska E, Gaston SM, Mamon H, Kulke MH, Zamponi R, Loda M, Maher E, Ogino S, Fuchs CS, Li J, Hader C, Makrigiorgos GM. (2007) DNA degradation test predicts success in whole-genome amplification from diverse clinical samples. J Mol Diagn. 9(4):441-51. [abstract]
von Ahlfen S, Missel A, Bendrat K, Schlumpberger M. (2007) Determinants of RNA quality from FFPE samples. PLoS ONE. 2(12):e1261. [abstract]
Doleshal M, Magotra AA, Choudhury B, Cannon BD, Labourier E, Szafranska AE. (2008) Evaluation and validation of total RNA extraction methods for MicroRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 10(3):203-11. [abstract]
LC Sciences internal research.
Li J, Smyth P, Flavin R, Cahill S, Denning K, Aherne S, Guenther SM, O'Leary JJ, Sheils O. (2007) Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells. BMC Biotechnol. 29;7:36. [abstract]
Hoefig KP, Thorns C, Roehle A, Kaehler C, Wesche KO, Repsilber D, Branke B, Thiere M, Feller AC, Merz H. (2008) Unlocking pathology archives for microRNA-profiling.Anticancer Res. 28(1A):119-23. [abstract]
Laios A, O'Toole S, Flavin R, Martin C, Kelly L, Ring M, Finn SP, Barrett C, Loda M, Gleeson N, D'Arcy T, McGuinness E, Sheils O, Sheppard B, O' Leary J. (2008) Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Mol Cancer. 7:35. [abstract]
Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K, Ju J. (2007) Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA. 13(10):1668-74. [abstract]
Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, Dienes HP, Odenthal M. (2008) MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology 47(4):1223-32. [abstract]

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